Capsid assembly and genome encapsidation are critical aspects in the life cycle of any virus. For the herpesviruses critical components of the machinery responsible for these processes have been identified by a combination of genetic and biochemical approaches. Much of this work has been done in the human pathogen, herpes simplex virus type 1 (HSV-1) since it is the most amenable to genetic and biochemical analysis, but mechanisms are likely to be shared by all herpesviruses. HSV-1 and HSV-2 are endemic in the population and are responsible for oral and genital infections, sight threatening infections and encephalitis. Our goal is to gain a better understanding of the processes by which head to tail concatemeric DMA molecules are taken up into preassembled capsids. In particular we will focus on the roles of two viral proteins: UL6, the portal protein and UL32 which may function as a viral chaperone needed for the formation of capsids which are competent for DMA uptake. We will also examine the protein-protein interactions between these proteins and UL15 and UL28 (putative terminase). In an exciting development, we have recently demonstrated that components of the capsid assembly and encapsidation machinery rely on host cell chaperones for proper folding and nuclear transport. When the cellular chaperone machinery is inhibited with drugs, viral.infection is blocked. This observation opens up a new avenue of antiviral drug discovery since cellular functions may provide a rich store of targets that can be exploited pharmacologically. In this proposal, an interdisciplinary approach involving genetics, molecular biology, biochemistry, cell biology, and biophysics will be used to further elucidate the role of viral and cellular chaperones in capsid assembly and encapsidation. Specific aims will test several hypotheses: 1) that host cell chaperones play a role in the assembly of capsids which are competent for cleavage and packaging; 2) that UL32 functions as a chaperone-like protein needed for proper localization and assembly of capsid components by facilitating disulfide bond formation; and 3) that UL6 plays a role in the assembly of competent capsids as well as cleavage and packaging through the stepwise assembly of the portal ring, incorporation of UL6 into growing capsids, and interaction with the terminase and viral DNA itself. Lay summary: The ability of a virus to package its genetic information in a stable protein coat (capsid) is essential for infectivity. In herpesviruses, the products of the DNA replication process are taken up into capsids by an active process involving several viral proteins. We have shown that host and viral helper proteins (chaperones) are needed for proper folding of these capsid components into a form that is competent to take up DNA. This grant will further our understanding of the mechanism by which this occurs and provide novel strategies for future antiviral therapies. -